Toxicology and Applied Pharmacology 247 (2010) 222–228
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Toxicology and Applied Pharmacology
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y t a a p
Effects of fluorotelomer alcohol 8:2 FTOH on steroidogenesis in H295R cells:
Targeting the cAMP signalling cascade
Chunsheng Liu a,b, Xiaowei Zhang c,⁎, Hong Chang d, Paul Jones d, Steve Wiseman d, Jonathan Naile d,
Markus Hecker d,e, John P. Giesy c,d,f, Bingsheng Zhou a,⁎
a
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
Graduate School of the Chinese Academy of Sciences, Beijing 100039, China
State Key Laboratory of Pollution Control and Resource Reuse & School of the Environment, Nanjing University, Nanjing, China
d
Department of Veterinary, Biomedical Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Canada
e
ENTRIX, Inc., Saskatoon, Saskatchewan, Canada
f
Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong SAR, China
b
c
a r t i c l e
i n f o
Article history:
Received 30 April 2010
Revised 18 June 2010
Accepted 28 June 2010
Available online 6 July 2010
Keywords:
Steroid hormone production
Gene expression
Adenylate cyclase
FTOH
a b s t r a c t
Previous studies have demonstrated that perfluorinated chemicals (PFCs) can affect reproduction by disruption of steroidogenesis in experimental animals. However, the underlying mechanism(s) of this
disruption remain unknown. Here we investigated the effects and mechanisms of action of 1H, 1H, 2H, 2Hperfluoro-decan-1-ol (8:2 FTOH) on steroidogenesis using a human adrenocortical carcinoma cell line
(H295R) as a model. H295R cells were exposed to 0, 7.4, 22.2 or 66.6 μM 8:2 FTOH for 24 h and productions
of progesterone, 17α-OH-progesterone, androstenedione, testosterone, deoxycorticosterone, corticosterone
and cortisol were quantified by HPLC-MS/MS. With the exception of progesterone, 8:2 FTOH treatment
significantly decreased production of all hormones in the high dose group. Exposure to 8:2 FTOH
significantly down-regulated cAMP-dependent mRNA expression and protein abundance of several key
steroidogenic enzymes, including StAR, CYP11A, CYP11B1, CYP11B2, CYP17 and CYP21. Furthermore, a dosedependent decrease of cellular cAMP levels was observed in H295R cells exposed to 8:2 FTOH. The observed
responses are consistent with reduced cellular cAMP levels. Exposure to 8:2 FTOH resulted in significantly
less basal (+ GTP) and isoproterenol-stimulated adenylate cyclase activities, but affected neither total
cellular ATP level nor basal (−GTP) or NaF-stimulated adenylate cyclase activities, suggesting that inhibition
of steroidogenesis may be due to an alteration in membrane properties. Metabolites of 8:2 FTOH were not
detected by HPLC-MS/MS, suggesting that 8:2 FTOH was not metabolized by H295R cells. Overall, the results
show that 8:2 FTOH may inhibit steroidogenesis by disrupting the cAMP signalling cascade.
© 2010 Elsevier Inc. All rights reserved.
Introduction
Perfluorinated chemicals (PFCs) are emerging pollutants and are
widely present in the environment, and in the blood of wildlife and
humans (Lau et al., 2007), which has generated scientific and regulatory
concern in terms of their potential health risk for humans and wildlife.
Recently, some PFCs have been shown to have potential endocrinedisruptive activities in vertebrates. For example, perfluorooctanoic acid
(PFOA) lowered concentrations of testosterone and cholesterol in serum
in rats (Biegel et al., 2001; Martin et al., 2007), and modified the mRNA
expression of genes associated with cholesterol biosynthesis and steroid
⁎ Corresponding authors. X. Zhang is to be contacted at School of the Environment,
Nanjing University, Nanjing, 210089, China. Fax: +86 25 83707304. B. Zhou, Institute of
Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China. Fax: +86 27
68780123.
E-mail addresses: howard50003250@yahoo.com (X. Zhang), bszhou@ihb.ac.cn
(B. Zhou).
0041-008X/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.taap.2010.06.016
metabolism in fetal mouse liver (Rosen et al., 2007). Exposure to
perfluorodecanoic acid (PFDA) decreased circulating concentrations of
testosterone and dihydrotestosterone in male rats (Bookstaff et al.,
1990). Furthermore, decreases in mRNA expression and the protein
abundance of steroidogenic enzymes were observed after acute and
chronic perfluorododecanoic (PFDoA) exposure in male rats. This
resulted in significantly lower concentrations of plasma testosterone,
compared to unexposed animals (Shi et al., 2007, 2009). These studies
suggest that PFCs can affect steroidogenic activities, however, the
cellular mechanisms remain unknown.
Fluorotelomer alcohols (FTOHs) belong to a class of telomerised
fluorinated chemicals and have been used for a variety of commercial
and industrial applications in paints, polishes, coatings, adhesives,
waxes, polymers, and electronics (Dinglasan-Panlilio and Mabury,
2006). Recently, these chemicals were found to have a wide
distribution in the ambient atmosphere (Jahnke et al., 2007; Stock
et al., 2004), including in indoor air and dust (Shoeib et al., 2006;
Strynar and Lindstrom, 2008). Previous studies have found that FTOHs
C. Liu et al. / Toxicology and Applied Pharmacology 247 (2010) 222–228
can be biotransformed to perfluorinated acids (Wang et al., 2005,
2009). Specifically, in both rat and mouse hepatocytes, 1H, 1H, 2H,
2H-perfluoro-decan-1-ol (8:2 FTOH) is metabolized to PFOA and
PFNA (Henderson and Smith, 2007; Martin et al., 2005), both of which
have been reported to occur in human and wildlife. Hence, FTOHbased substances have been proposed as possible indirect sources
for some PFC exposure. Furthermore, exposure to 1H, 1H, 2H, 2Hperfluorooctan-1-ol (6:2 FTOH) or 8:2 FTOH significantly altered
plasma concentrations of testosterone and estradiol in zebrafish
through effects on the hypothalamic–pituitary–gonadal (HPG) axis
(Liu et al., 2009a,b). However, the cellular mechanisms underlying
such steroidogenic disruption has remained unclear.
The present study investigated the mechanism(s) of alteration of
steroidogenic functions by 8:2 FTOH in H295R human adrenocortical
carcinoma cells. H295R cells retain physiological characteristics of
zonally undifferentiated fetal adrenal cells and express all the key
enzymes of steroidogenesis (Fig. 1). Upon cAMP stimulation, these cells
produce all the adrenal steroid hormones found in the adult adrenal
cortex (Gazdar et al., 1990; Sanderson, 2006). H295R cells have been
employed for studying the biological effects and mechanisms of action
of a variety of chemicals (e.g., Ding et al., 2007; Hecker et al., 2006, 2007;
Hilscherova et al., 2004; Li and Wang, 2005; Sanderson, 2006; Zhang
et al., 2005). In the present study, HPLC-MS/MS was used to examine
the effects of 8:2 FTOH on the production of seven steroid hormones:
progesterone, 17α-OH-progesterone, androstenedione, testosterone,
deoxycorticosterone, corticosterone and cortisol. Real-time quantitative
polymerase chain reaction (qRT-PCR) and Western blotting analyses
were employed to characterize alterations in mRNA expression and
protein abundance, respectively, of steroidogenic enzymes. In addition,
cellular cAMP and ATP levels, and basal adenylate cyclase activity were
measured. Basal activity, independent of G-proteins, was also measured
to assess expression and constitutive adenylate cyclase activity. In order
to evaluate the roles of interactions between 8:2 FTOH and G-protein
or membrane receptors, the basal activities in the presence of NaF or
isoproterenol, which elicit maximal G-protein and β-adrenergic
stimulation, respectively, were assessed.
Materials and methods
Chemicals. 1H, 1H, 2H, 2H-perfluoro-decan-1-ol (8:2 FTOH) (purity
N97%) was obtained from Strem Chemicals (Newburyport, MA, USA).
Surrogate deuterated standards, progesterone-d9, 17α-OH-progester-
223
one-d8, androstenedione-d7, testosterone-d5, deoxycorticosteroned8, were provided by C/D/N Isotope (Pointe-Claire, Quebec, Canada).
Cortisol-d4 was provided by Cambridge (Andover, MA, USA). 3[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was
obtained from Duchefa (Haarlem, the Netherlands). All other reagents
used were obtained from Sigma (St. Louis, MO, USA).
Cell culture and chemicals exposure. The H295R human adrenocortical carcinoma cell line was obtained from the American Type Culture
Collection (ATCC, Beltsville, VA, USA) and was cultured in DMEM/F12
medium (Sigma), containing 1% insulin-transferring sodium selenite
plus Premix (ITS) (BD Bioscience, Bedford, USA) and 2.5% Nu-Serum
(BD Bioscience) at 37 °C in a 5% CO2 atmosphere. For chemical
exposure, H295R cells were seeded in different size of plates
depending on the type of assays. The medium volume of every well
for 96-well, 24-well and 6-well plate was 0.2, 1.0 and 3.0 mL,
respectively. After culture for 24 h, cells were exposed to 0, 7.4, 22.2
or 66.6 μM 8:2 FTOH dissolved in dimethyl sulfoxide (DMSO; Sigma)
for 24 h. Control treatments received 0.1% DMSO (v/v). The exposure
concentrations were chose based on the information from previous
studies (Lau et al., 2007; Martin et al., 2007).
Cell viability assay. Cell viability was determined by measuring MTT
activity using previous method (Hansen et al., 1989). Briefly, H295R
cells were seeded into 24-well plates (Corning Life Sciences, Corning,
NY, USA) at a density of 3 × 105 cells/ml. After culture for 24 h, the
cells were exposed to 0, 7.4, 22.2 and 66.6 μM 8:2 FTOH for 24 h, then
the culture medium was removed and MTT activity was determined.
Four replicates were used in each experiment.
Quantification of steroid hormones. For the quantification of steroid
hormone concentrations in culture medium samples were extracted by
ethyl acetate/hexane (v/v, 50/50) followed by LC-MS/MS analysis.
Surrogate deuterated standards, progesterone-d9, 17α-OH-progesterone-d8, androstenedione-d7, testosterone-d5, deoxycorticosteroned8, and cortisol-d4, were spiked into samples before the extraction.
For all steroid hormones, chromatography was performed using nanopure water and methanol. Sample extracts were separated on a Betasil
C18 (100× 2.1 mm, 5 μm particle size) column from Thermo (Waltham,
MA, USA) and then analyzed using an ABI SCIEX 3000 triple quadrapole
tandem mass spectrometer (ABI SCIEX, Milford, MA, USA). All data
were acquired and processed with AB Sciex Analyst 1.4.1 software
Fig. 1. Steroidogenic pathway in the H295R cells.
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C. Liu et al. / Toxicology and Applied Pharmacology 247 (2010) 222–228
(Applied Bioscience, Foster City, CA, USA). Three replicates were used
in each experiment.
Western blotting analysis.
Western blotting analysis was performed
as previously described (Yu et al., 2008; Zhang et al., 2009) with some
modifications. Briefly, cells were seeded in 6-well plates (Corning Life
Sciences). After exposure, cells were lysed and the protein content
was determined. 50 μg cytoplasmic protein were denatured, electrophoresed and transferred onto a polyvinylidene difluoride (PVDF)
membrane. The transferring efficiency was evaluated for equal
protein in each lane using a reversible dye (PIERCE, IL, USA). The
membrane was blocked and blots were probed with an anti-human
steroidogenic acute regulatory protein (StAR), side-chain cleavage
enzyme (CYP11A), steroid 11β hydroxylase (CYP11B1), aldosterone
synthetase (CYP11B2), steroid 17α-hydroxylase/17, 20-lyase
(CYP17) and steroid 21-hydroxylase (CYP21) antibodies (Santa Cruz
Biotechnology Inc., Santa Cruz, CA, USA). Following primary antibody
incubation the membrane was washed and incubated with either a
horseradish peroxidase-conjugated anti-mouse or anti-goat antibody
depending on the origin of the primary antibodies (Santa Cruz
Biotechnology Inc.). Each secondary antibody was diluted 1:2000 in
skim milk blocking solution. The immunoblot analysis was performed
using the Amershanm™ ECL Plus Western Blotting Detection System
(GE Healthcare, Baie-d'Urfe, QC, Canada). The quantification of
the relative expression of steroidogenic enzymes was performed
by using BandScan 5.0 software. Three replicates were used in each
experiment.
Quantitative real-time PCR assay. Briefly, cells were seeded in 24well plates (Corning Life Sciences). After exposure, the culture
medium was removed for further hormone analysis and cells were
used for total RNA isolation (Agilent Technologies, Santa Clara, CA,
USA). The RNA purity was determined by measuring the 260/280 nm
ratios and 1% agarose-formaldehyde gel electrophoresis with
ethidium bromide staining. The purified RNA was used immediately
for reverse transcription or stored at −80 °C until analysis. Firststrand cDNA synthesis was performed using an Iscript™ cDNA
synthesis kit (BioRad, Mississauga, ON, Canada) with a 1 μg aliquot
of cellular total RNA. Quantitative real-time PCR (qRT-PCR) was
analyzed on the ABI 7300 real time PCR system (Applied Biosystems)
using SYBR® Green PCR master mix (Applied Biosystems). The primer
design and concentrations, thermal cycle profiles and quantification
of target genes were performed as described previously (Zhang
et al., 2005). The housekeeping gene prophobilinogen deaminase
(PBGD) did not vary under experimental conditions in the present
study (data not shown) and was used as an internal control. Gene
expression was measured in triplicate and this experiment was
repeated three times.
gation steps (1000g for 5 min and 40,000g for 20 min at 4 °C) and the
protein content was determined. 50 μg of membrane protein was used
for each assay. Adenylate cyclase activity was determined in four
different ways. First, basal activity was evaluated by incubating the
membrane protein for 15 min at 37 °C in reaction buffer (50 mM Tris–
HCl (PH 7.4), 5 mM MgCl2, 0.5 mM EDTA, 1 mM ATP, 0.1 μM GTP,
0.2 IU pyruvate kinase, 0.1 U myokinase and 2.5 mM phosphoenolpyruvate). Second, basal activity independent of G-protein was
determined to assess expression and constitutive adenylate cyclase
activity by measuring the basal activity in the absence of GTP. Third,
the net G-protein-linked response was assessed by measuring
the basal activity in the presence of 10 mM NaF, which elicits maximal
G-protein stimulation. Finally, receptor-targeted effects were evaluated by measuring the basal activity in the presence of 100 μM
isoproterenol, which elicits maximal β-adrenergic stimulation. Three
replicate wells were used in each experiment.
Determination of metabolites of 8:2 FTOH. H295R cells were seeded
into 24-well plates (Corning Life Sciences) at a concentration of
3 × 105 cells/ml. After exposure for 24 h, the cells and medium were
collected for metabolites extraction using previous method with some
modifications (Yeung et al., 2006), followed by HPLC-MS/MS analysis.
Briefly, the sample and 2 mL of methyl-tert-butyl ether (MTBE)
were added to a polypropylene tube, and the mixture was shaken for
20 min and centrifuged for 15 min at 3000g. The organic layer was
collected. Sample extracts were separated on a Betasil C18 column from
Thermo (Waltham, MA, USA) and then analyzed using an ABI SCIEX
3000 triple quadrapole tandem mass spectrometer (ABI SCIEX, Milford,
MA, USA). All data were acquired and processed with AB Sciex Analyst
1.4.1 software (Applied Bioscience, Foster City, CA, USA). A total of 12
PFCs were identified and quantified by comparison to authentic
standards. These metabolites included perfluorobutanesulfonate
(PFBS), perfluorohexanesulfonate (PFHS), perfluorooctanesulfonate
(PFOS), perfluoro-n-butyric acid (PFBA), perfluoropentanoic acid
(PFPnA), perfluoroheptanoic acid (PFHA), perfluoroheptanoic acid
(PFHpA), perfluorooctanoate (PFOA), perfluorononanoic acid (PFNA),
perfluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFuDA),
perfluorododecanoic acid (PFDoDA). The detection limit for all of
these compounds was 200 ng/L. Three replicate wells were used in each
experiment.
Statistical analysis. Normality and homogeneity of variances of
each parameter were determined by the Kolmogorov–Smirnov and
Levene's test, respectively. One-way analysis of variance (ANOVA)
and Tukey's multiple range tests were used to determine significant
differences between control and exposure treatments. The criterion
for statistical significance was p b 0.05.
Results
Quantification of ATP and cAMP. Cellular ATP and cAMP were determined using commercial kits following the manufacturer's
specifications. Briefly, for the ATP assay, H295R cells were grown in
96-well plates (Corning Life Sciences) for 8:2 FTOH exposure. After
exposure, the medium was removed and the cells were used for
ATP content detection using a kit (ViaLight® Plus Proliferation and
Cytotoxicity BioAssay Kit, Lonza, Rockland, ME, USA). For the cAMP
assay, cells were seeded in 6-well plates (Corning Life Sciences). After
exposure, the medium was removed and cells were used to determine
the cAMP content using a cAMP enzyme immunoassay kit (Sigma).
Three replicate wells were used in each experiment.
Adenylate cyclase activity. Adenylate cyclase activity was determined using the protocol described in earlier studies (Liu et al.,
2005; Song et al., 1997). Briefly, cells were plated in 100 × 20 mm
Petri dishes. After 8:2 FTOH exposure, cells were collected and
homogenized. Membranes were obtained by two different centrifu-
H295R cell viability
No significant change in cell viability was observed in any of the
8:2 FTOH treatments after 24 h as indicated by MTT activity (data
not shown).
Effects on production of the steroid hormones
No statistically significant effects on progesterone production
were observed after 22.2 or 66.6 μM 8:2 FTOH exposure compared
with the control (Table 1). No significant change in production of any
of the steroid hormones was observed in cells exposed to 7.4 μM 8:2
FTOH. Only testosterone production, which was decreased by 18.6%,
was affected by exposure to 22.2 μM 8:2 FTOH. However, exposure to
66.6 μM of 8:2 FTOH significantly inhibited production of 17α-OHprogesterone, androstenedione, testosterone, deoxycorticosterone,
C. Liu et al. / Toxicology and Applied Pharmacology 247 (2010) 222–228
225
Table 1
Steroid hormone concentrations (mean ± S.E.M.) in H295R cells exposed to various
concentrations of 8:2 FTOH for 24 h.
Steroid hormones
8:2 FTOH concentrations
0 mg/L
7.4 μM
22.2 μM
66.6 μM
Progesterone (ng/mL)
0.74 ± 0.10
0.71 ± 0.04
0.66 ± 0.03
0.69 ± 0.12
17α-OH-Progesterone
6.12 ± 0.09
5.94 ± 0.32
5.52 ± 0.23
4.56 ± 0.27*
(ng/mL)
Androstenedione
35.20 ± 2.60 31.73 ± 1.12 31.07 ± 1.00 18.87 ± 3.65*
(ng/mL)
Testosterone (ng/mL)
0.43 ± 0.01
0.40 ± 0.04
0.35 ± 0.01*
0.16 ± 0.05*
Deoxycorticosterone
38.90 ± 0.10 36.73 ± 0.33 30.05 ± 6.17 24.10 ± 0.40*
(ng/mL)
Corticosterone
21.33 ± 0.53 19.86 ± 0.97 16.67 ± 4.15 11.73 ± 2.37*
(ng/mL)
Cortisol (ng/mL)
4.93 ± 0.20
4.77 ± 0.17
4.69 ± 0.05
2.54 ± 0.31*
*Pb 0.05 indicates significant difference between exposure groups and the corresponding
control group.
corticosterone and cortisol by 25.5%, 46.4%, 62.8%, 38.1%, 45.0%, and
48.5%, respectively (Table 1).
Effects on mRNA expression and protein abundance of the steroidogenic
enzymes
Exposure to 22.2 or 66.6 μM 8:2 FTOH significantly down-regulated
StAR, CYP11A, CYP11B1, CYP11B2 and CYP21 mRNA expression by
15.0% and 35.1%, 17.1% and 18.8%, 17.2% and 53.5%, 24.7% and 35.7%,
and 26.2% and 34.8%, respectively. In contrast, CYP17 mRNA
expression was down-regulated by 22.3% only in the 66.6 μM exposure
groups compared with the control (Fig. 2). No significant effects on
mRNA expression of 3βHSD (3β-hydroxysteroid dehydrogenase
isomerase) or 17βHSD (17β-hydroxysteroid dehydrogenase) were
observed after 8:2 FTOH exposure at all the tested concentrations
compared with the control (Fig. 2). Exposure to 22.2 or 66.6 μM 8:2
FTOH significantly down-regulated CYP11A and CYP21 protein
expression by 40.2% and 65.2%, 54.9% and 54.3%, respectively. StAR,
CYP11B1, CYP11B2 and CYP17 protein expressions were downregulated by 42.4%, 51.0%, 43.3% and 44.7% only in the 66.6 μM
exposure groups, respectively, compared with the control (Fig. 3).
Fig. 3. Abundance of steroidogenic enzymes in H295R cells exposed to 0, 7.4, 22.2 or
66.6 μM 8:2 FTOH for 24 h. (A) Representative Western blot of steroidogenic enzymes
from control and 8:2 FOH-exposed cells; (B) Quantification of the relative expression of
steroidogenic enzymes in control and treatment groups. Values represent mean ± S.E.
M. of three replicate samples. Significant differences (p b 0.05) between control and
exposure groups are indicated by an asterisk (*).
Effects on cellular cAMP and ATP contents and adenylate cyclase activity
Exposure to 22.2 or 66.6 μM 8:2 FTOH significantly decreased
cellular cAMP levels by 28.4% and 46.0%, respectively, compared with
the control (Fig. 4). No statistically significant effect on cellular ATP
content after 8:2 FTOH exposure was detected at any of the tested
concentrations (data not shown). Exposure to 66.6 μM 8:2 FTOH
significantly depressed basal adenylate cyclase activity by 27.6%,
whereas the activity remained unchanged at the lesser concentrations
(7.4 or 22.2 μM) (Table 2). Similarly, statistically significant decreases
(19.4%) were observed in isoproterenol-stimulated adenylate cyclase
activity after 66.6 μM 8:2 FTOH exposure, whereas no significant
change in this enzyme activity was observed in cells exposed to 7.4
Fig. 2. mRNA expression of steroidogenic genes in H295R cells exposed to 0, 7.4, 22.2 or 66.6 μM 8:2 FTOH for 24 h. Values represent mean ± S.E.M. of three replicate samples.
Significant (p b 0.05) differences between control and exposure groups are indicated by an asterisk (*).
226
C. Liu et al. / Toxicology and Applied Pharmacology 247 (2010) 222–228
Fig. 4. Cellular cAMP levels in the H295R cells exposed to 0, 7.4, 22.2 or 66.6 μM 8:2
FTOH for 24 h. Values represent mean ± S.E.M. of three replicate samples. Significant
differences (p b 0.05) between control and exposure groups are indicated by an asterisk (*).
or 22.2 μM 8:2 FTOH (Table 2). Exposure to 8:2 FTOH did not
significantly affect basal activity independent G-protein and NaFstimulated activity compared with the control (Table 2).
Metabolite measurement
In the current study, 12 metabolites have not been observed in 8:2
FTOH treated H295R cells.
Discussion
In the present study, a significant inhibition of steroidogenesis was
observed in H295R cells exposed to 8:2 FTOH, as demonstrated by the
decreased production of the steroid hormones, 17α-OH-progesterone, androstenedione, testosterone, deoxycorticosterone, corticosterone and cortisol. The mechanisms of the inhibitory effects involved
down-regulation of StAR, CYP11A, CYP11B1, CYP11B2, CYP17 and
CYP21 mRNA expression and protein abundance. In addition, the
down-regulation of mRNA expression and protein abundance of key
steroidogenic enzymes was accompanied by decreased cellular cAMP.
Furthermore, our results suggest that the decreased cAMP content
was due to reduced membrane receptor-mediated adenylyl cyclase
activities and not due to depressed cellular ATP concentrations.
Exposure to fluorotelomer alcohols (FTOHs) has been implicated
as a possible pathway of exposure that results in PFCs accumulation in
humans and wildlife. Previous studies reported that certain PFCs, such
as PFOA, PFDA, and PFDoA decreased serum testosterone level in rats
under toxicological dosages (Bookstaff et al., 1990; Martin et al., 2007;
Table 2
Adenylate cyclase activity (mean ± S.E.M.) in H295R cells exposed to various concentrations of 8:2 FTOH for 24 h.
Adenylate
cyclase
activitiesa
8:2 FTOH concentrations
Basal activity
(+GTP)
Basal activity
(−GTP)
NaF-stimulated
activity
Isoproterenolstimulated
activity
135.18± 13.21
143.48 ± 11.59 125.42 ± 14.03
97.81 ± 2.02*
104.54 ± 3.85
114.75 ± 8.94
97.74 ± 5.30
0 μM
7.4 μM
22.2 μM
118.03 ± 7.87
66.6 μM
818.65± 66.14 1007.59 ± 63.30 779.61± 104.03 777.54 ± 66.11
179.60 ± 8.91
190.22 ± 12.12 184.86 ± 8.11
144.74 ± 1.25*
*Pb 0.05 indicates significant difference between exposure groups and the corresponding
control group.
a
Adenylate cyclase activities were expressed as pmol cAMP/min/mg protein.
Shi et al., 2007, 2009). The serum PFCs concentrations (5 μg/mL to
260 μg/mL) measured in those dosed animals were in the same range
as the concentrations of FTOHs applied to H295R cells in this study
(Lau et al., 2007; Martin et al., 2007). It has been shown that exposure
to 6:2 FTOH or 8:2 FTOH significantly disrupted plasma concentrations of sex steroids in zebrafish with a LOEC of 30 μg/L for both
chemicals (Liu et al., 2009a,b). The present study further expanded on
investigating the underlying mechanisms of this inhibitory effect:
exposure to 8:2 FTOH significantly decreased 17α-OH-progesterone,
androstenedione and testosterone production by H295R cells,
whereas no effect on progesterone production was observed. These
results suggested that the reduction in testosterone level reported
previously might partially result from decreased production of upstream hormones (e.g., 17α-OH-progesterone, androstenedione). In
addition, exposure to 8:2 FTOH in H295R cells also caused significant
decreases in deoxycorticosterone, corticosterone and cortisol production. These hormones are associated with a variety of physiological
processes including metabolism, stress responses, immune responses,
vasoconstriction, growth, development, salt and water homeostasis
and vascular tone (Mommsen et al., 1999; Osborn, 1991). Studies
have shown that decreased production of these hormones may impair
normal physiological processes and cause pathologic effects in vivo
(Peter et al., 1999; Ueshiba et al., 2002).
This study further examined whether the inhibitory effects of 8:2
FTOH on steroidogenesis resulted from the down-regulation of
mRNA expression and the consequent protein abundance of
steroidogenic enzymes. Exposure to 8:2 FTOH significantly downregulated StAR, CYP11A, CYP11B1, CYP11B2, CYP17 and CYP21
mRNA expression, and protein abundance in a dose-dependent
manner. These enzymes play key roles in steroid hormone
biosynthesis (Fig. 1). Therefore, the down-regulation of mRNA
expression and protein abundance could lead to the decreased
steroid hormone production. It has been previously demonstrated
that exposure to PFDoA significantly decreases StAR and P450scc
(CYP11A) mRNA expression and protein abundance resulting in
decreased serum testosterone concentrations in male rat testes (Shi
et al., 2007, 2009). The authors speculated that decreased testosterone level, at least in part, was due to the reduced expression of StAR
and CYP11A. On the other hand, we did not observe a significantly
inhibitory effect on progesterone production. Progesterone biosynthesis is catalyzed by 3βHSD using pregnenolone as substance, and
then is converted to 17α-OH progesterone and 11-deoxycorticosterone by CYP17 and CYP21, respectively (Fig. 1). In the present study, 8:2
FTOH did not affect 3βHSD mRNA expression, but significantly downregulated CYP17 and CYP21 mRNA expression and protein abundance.
Therefore, the down-regulation of CYP17 and CYP21 mRNA expression
and protein abundance could result in accumulation of progesterone.
That is a possible reason for unchanged progesterone production after
8:2 FTOH treatment despite the down-regulation of StAR and CYP11A
mRNA expression and protein abundance, which could result in less
pregnenolone, the precursor of progesterone. Taken together, the
observation that decreased mRNA expression was associated with a
decrease in protein abundance suggests that 8:2 FTOH acts at the level
of gene transcription.
cAMP is an important secondary messenger that stimulates steroid
hormone biosynthesis in the human adrenal cortex (Sewer and
Watermen, 2001; Stocco et al., 2005). The transcriptional profiles of
most steroidogenic genes (e.g., StAR, CYP11A, CYP11B, CYP17 and
CYP21) are cAMP-dependent (Sewer and Watermen, 2001). Many
chemicals have been hypothesized to disrupt steroidogenesis via
the cAMP pathway, including triazines, forskolin, vinclozolin, isobutyl,
and methylxanthine (Hilscherova et al., 2004; Sanderson et al.,
2000, 2002). To test the hypothesis that 8:2 FTOH exposure inhibits
steroidogenesis (including decreased mRNA expression, protein
abundance and hormones levels) by reducing cellular cAMP levels,
cellular cAMP content was examined. In cells exposed to 22.2 or
C. Liu et al. / Toxicology and Applied Pharmacology 247 (2010) 222–228
66.6 μM 8:2 FTOH cAMP content was significantly decreased by 28.4%
and 45.9%, respectively, which indicates that the decrease in cellular
cAMP may lead to inhibition of steroidogenesis. This result is
consistent with those of the previous study reporting that a 50%
decrease in cellular cAMP level significantly reduced aldosterone
production by approximately 75% in bovine adrenal glomerulosa cells
treated with atrial natriuretic peptide (ANP) (MacFarland et al., 1991).
Adenylate cyclase is the enzyme responsible for catalyzing
conversion of cellular ATP to cAMP via a G-protein-coupled receptor
pathway (Stocco et al., 2005). In the present study, the decreased
basal adenylate cyclase activity and absence of effect on ATP levels
suggest that 8:2 FTOH could decrease cellular cAMP by depressing
basal adenylate cyclase activity. To further investigate the mechanism
(s) of 8:2 FTOH effects on adenylate cyclase, we assessed the
constitutive, NaF-stimulated, and isoproterenol-stimulated activity
of adenylate cyclase in 8:2 FTOH exposed cells. 8:2 FTOH exposure
significantly depressed the isoproterenol-stimulated adenylate cyclase activity, but no effects on constitutive activity and NaFstimulated activity were observed. These results suggested that 8:2
FTOH could affect a membrane G-protein-coupled receptor pathway
by changing membrane properties and thus depress membrane
receptor-stimulating adenylate cyclase activity. As stated above, 8:2
FTOH can be metabolized to PFOA and PFNA in rat and mouse
hepatocytes. To further verify whether 8:2 FTOH could be metabolized
and possible effects from these metabolites on H295R cells, we also
measured 12 metabolites. However, in the present study, none of
these metabolites were detected in H295R cells and culture medium,
indicating that 8:2 FTOH was not metabolized by the cells. These
results suggested that the inhibitory effects on steroidogenesis were
affected by 8:2 FTOH, not its metabolites.
In summary, we hypothesize that 8:2 FTOH has the potential to
depress steroidogenesis (including decreased mRNA expression,
protein abundance, and hormone levels) through reduced cellular
cAMP levels. Inhibition of cAMP biosynthesis by adenylate cyclase as a
result of interaction with membrane receptors appears to be the most
likely mechanism for decreased cAMP levels. This study clearly
underlines the need for in vivo studies to examine the accumulation
and disposition of 8:2 FTOH in the body as well as determining effects
on steroidogenesis. Furthermore, it would be useful to determine
whether metabolites of 8:2 FTOH or the compound itself are
responsible for the observed effects on steroidogenesis.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgments
This study was supported by the National Natural Science Foundation of China (20890113, 20877094). The research was also supported
by a Discovery Grant from the NSERC of Canada (Project #326415-07)
and a grant from the WED Canada (Project #6578 and 6807) to the
University of Saskatchewan.
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